Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice

Abstract

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children, but no vaccine is currently available. Live-attenuated vaccines represent an attractive immunization approach; however, balancing attenuation while retaining sufficient immunogenicity and efficacy has prevented the successful development of such a vaccine. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. The M protein is required for virion assembly following infection of a host cell but is not necessary for either genome replication or gene expression. Therefore, infection with RSV M-null produces all viral proteins except M but does not generate infectious virus progeny, resulting in a single-cycle infection. We evaluated RSV M-null as a potential vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared with its recombinant wild-type control virus (RSV recWT). RSV M-null-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared with mice infected with RSV recWT. Despite its attenuation, RSV M-null infection induced robust immune responses of similar magnitude to that elicited by RSV recWT. Additionally, RSV M-null infection generated serum Ab and memory T cell responses that were similar to those induced by RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, our results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

Figure 1.. No infectious progeny is detected following RSV M-null infection in vivo.

(A) Diagram depicting the genome content of RSV recWT and RSV M-null. (B-E) BALB/c mice were infected with either RSV recWT or RSV M-null, and lungs were harvested on days 1, 2, 4, and 7 p.i. (B) EGFP⁺ focus-forming units and (C) infectious PFU were quantified in the lung by plaque assay using H2-M helper cells. Open (recWT) and closed (M-null) circles represent values for individual mice and lines indicate mean ± SEM of 2 independent experiments (n=8). (D) RSV N gene and (E) RSV M gene copy numbers per lung were determined by real-time PCR. Data are presented as mean ± SEM of representative results from 1 of 2 independent experiments (n=4). Dashed lines denote the limit of detection for each assay. Groups were compared using one-way ANOVA, ** p<0.01, *** p<0.001.

Figure 2.. RSV M-null infection reduces weight loss and pulmonary dysfunction.

BALB/c mice were infected with either RSV recWT or RSV M-null and monitored daily for (A) weight loss and respiratory parameters including (B) Penh, (C) EF50, and (D) respiratory rate by whole body plethysmography. Data are presented as mean ± SEM of 6 independent experiments (n=35 for recWT and n=36 for M-null). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 3.. RSV M-null infection induces T cell responses of similar magnitude to recWT infection.

BALB/c mice were infected with either RSV recWT or RSV M-null, and lungs were harvested on days 6, 8, and 10 p.i. Total numbers of (A) CD11a^hiCD49d⁺ CD4 T cells, (B) CD11a^hiCD8^lo CD8 T cells, and (C) M2₈₂-, (D) M2₁₂₇-, (E) F₈₅-, and (F) F₂₄₉-tetramer⁺ CD8 T cells were quantified. Total numbers of (G) CD4 T cells and (H) CD8 T cells producing IFN-γ after re-stimulation with G₁₈₃ and M2₈₂ peptides, respectively, were determined by intracellular cytokine staining. Data are presented as mean ± SEM of 2–3 independent experiments (n=8 for days 6 and 10 and n=12 for day 8). Groups were compared using Student’s t test, ** p<0.01.

Figure 4.. T follicular helper cells are generated by RSV M-null infection.

BALB/c mice were infected with either RSV recWT or RSV M-null, and mLN and lungs were harvested on days 14 and 21 p.i. Cells were gated on CD4 T cells, and gates were placed using fluorescence minus one controls. Representative staining panels of Bcl6⁺CXCR5⁺ Tfh cells in the (A) mLN and (C) lung. Total numbers of Bcl6⁺CXCR5⁺ Tfh cells in the (B) mLN and (D) lung. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using Student’s t test.

Figure 5.. RSV M-null infection induces similar numbers of germinal center B cells as recWT infection.

BALB/c mice were infected with either RSV recWT or RSV M-null, and mLN and lungs were harvested on days 14 and 21 p.i. Total numbers of CD19⁺ B cells in the (A) mLN and (B) lung. Representative staining panels of Fas⁺GL-7⁺ GC B cells in the (C) mLN and (E) lung. Total numbers of Fas⁺GL-7⁺ GC B cells in the (D) mLN and (F) lung. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using Student’s t test, * p<0.05.

Figure 6.. RSV M-null infection generates long-lasting RSV-specific serum antibody responses.

BALB/c mice were infected with either RSV recWT or RSV M-null, and serum was collected on days 14, 28, and 84 p.i. Serum was assessed for levels of RSV-specific (A) total Ig, (B) IgG1, and (C) IgG2a by ELISA. Data are presented as mean ± SEM of representative results from 1 of 2 independent experiments (n=5). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 7.. RSV M-null infection induces RSV-specific tissue-resident memory CD8 T cells.

BALB/c mice were infected with either RSV recWT or RSV M-null and administered anti-CD45 antibody intravascularly 3 minutes prior to euthanasia on days 30 and 100 p.i. Cells negative for staining with the CD45 intravascular antibody (IV⁻) were gated, and total numbers of (A) CD11a^hiCD49d⁺ CD4 T cells, (B) CD11a^hiCD8^lo CD8 T cells, and (C) M2₈₂-tetramer⁺ CD8 T cells within the lung parenchyma are shown. (D) Representative staining panels and (E) total numbers of CD69⁺CD103⁺ IV⁻ M2₈₂-tetramer⁺ CD8 T cells. Data are presented as mean ± SEM of 2 independent experiments (n=10 for day 30 and n=9 for day 100). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 8.. RSV M-null infection provides protection against secondary viral challenge.

(A/B) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with RSV either (A) 6 or (B) 12 weeks p.i. Lung RSV titers on day 4 p.i. were determined by plaque assay using VERO cells. (C/D) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with IAV-M2₈₂ either (C) 6 or (D) 12 weeks p.i. Lung IAV titers on day 6 p.i. were determined by plaque assay using MDCK cells. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using one-way ANOVA, *** p<0.001.